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Sonoporation: Mechanistic Insights and Ongoing Challenges for Gene Transfer Anthony Delalande, Spiros Kotopoulis, Michiel Postema, Patrick Midoux, Chantal Pichon

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Sonoporation: Mechanistic Insights and Ongoing Challenges for Gene Transfer Anthony Delalande, Spiros Kotopoulis, Michiel Postema, Patrick Midoux, Chantal Pichon Sonoporation: mechanistic insights and ongoing challenges for gene transfer Anthony Delalande, Spiros Kotopoulis, Michiel Postema, Patrick Midoux, Chantal Pichon To cite this version: Anthony Delalande, Spiros Kotopoulis, Michiel Postema, Patrick Midoux, Chantal Pichon. Sonopo- ration: mechanistic insights and ongoing challenges for gene transfer. Gene, Elsevier, 2013, 525 (2), pp.191-199. 10.1016/j.gene.2013.03.095. hal-03195625 HAL Id: hal-03195625 https://hal.archives-ouvertes.fr/hal-03195625 Submitted on 11 Apr 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Sonoporation: mechanistic insights and ongoing challenges for gene transfer Anthony Delalande1, Spiros Kotopoulis2, Michiel Postema2, Patrick Midoux1, Chantal Pichon1* 1. Centre de Biophysique Moléculaire, CNRS, Orléans, France. 2. Department of Physics and Technology, University of Bergen, Bergen, Norway. * Corresponding author: [email protected] telephone: +33 238255595 fax: +33 238631517 Abstract Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drugs and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed. We also outline ongoing challenges for clinical applications. Key words: ultrasound; microbubbles; physical gene delivery method; gene therapy 1 2 1. Introduction The principle of gene therapy is to introduce gene or nucleic acids into cells to cure genetic deficiencies. The success of gene therapy obtained with the use of viral vectors demonstrates unambiguously the feasibility of this innovative therapy [1-3]. To date, viral vectors remain the best vehicles to introduce genes into cells. Nevertheless, there are still drawbacks inherent to the use of viral molecule observed in gene therapy clinical trials raising serious safety concerns [4, 5]. In addition, size limitation capacity, cell targeting and manufacturing issues are still difficult to handle despite tremendous progresses made on viral vector bioengineering. Therefore, there is still some room for the development of alternative approaches of high safety, low immunogenicity and easy manufacture. This last decade, many efforts have been done to search for non-viral options. The goal is to design synthetic gene delivery systems that incorporate viral-like features to transfect efficiently cells [6-8]. Among non-viral systems, chemical vectors are the most widely used. These vectors have to face on several extracellular and cellular barriers to reach efficiently the target cells. One of the main challenges is the lack of selectivity towards target tissues explaining their narrow therapeutic index. Lack of specificity causes high toxicity which hampers the efficacy at the target site. For that, designing an efficient targeted delivery strategy is of importance to further improve the delivery systems while reducing side effects. To target chemical vectors, it is possible to couple them with ligands specific to receptors present at the cellular surface of target cells. A second option is to use an externally applied trigger to control the gene delivery in the targeted area. These two strategies are not mutually exclusive and could be combined. Physical trigger can be used either alone or combined with chemical or viral vectors to improve the targeting and/or gene expression efficiency. There are several physical methods 3 starting from hydrodynamic injection to more sophisticated systems such as electroporation-based on electric fields or ultrasound-mediated delivery. 2. Ultrasound as physical method for delivery Ultrasound can be used for imaging (ultrasonography) and for physical therapy (pulsed ultrasound mode) [9, 10]. These last years, therapeutic applications of ultrasound have gained new interests as a result of its exploitation for drug or gene delivery. Depending on the energy delivered by ultrasound, two types of effects can be produced either thermal or non-thermal each of them having their own application. High ultrasound intensities produce heating due to the absorption of acoustic energy by tissues; this property is employed in high-intensity focused ultrasound (HIFU) surgery or ultrasound-based physiotherapy. "World Federation for Ultrasound in Medicine and Biology Temperature" has stated that an elevation of 1.5°C is considered safe while an elevation of 4-5°C during 5 minutes could be dangerous [11]. At low ultrasound intensities, cavitation and mechanical streaming are the main non-thermal effects obtained. These effects can induce some benefits as tissue healing or ultrasound-mediated delivery. Inertial cavitation is the process of formation, oscillation and collapse of gaseous bubbles driven by an acoustic field [24]. The presence of preformed microbubbles in the environment allows reducing the threshold of energy needed for cavitation. Used as external trigger, ultrasound permits to spatiotemporally control the release of drug encapsulated in microbubbles or in their surrounding in a non-invasive manner [12-18]. 4 3. Microbubbles and sonoporation Microbubbles are gas-filled particles consisting of a gas core encapsulated by a stabilizing shell. They have been first developed as ultrasound contrast agents to differentiate blood and their surroundings under ultrasound due to their low acoustic impedance difference. When microbubbles are driven by ultrasound at a frequency close to their resonance frequency, they oscillate and produce sound [19, 20]. These oscillations lead to an increased permeability of surrounding cells allowing a targeted local drug delivery. The increased cellular uptake has been attributed to the formation of transient pores in the cell membrane facilitating trans-membrane transport of drugs into the cell [21-24]. This transient permeabilization of a cell membrane is called sonoporation. 3.1. Microbubble-cell interactions Microbubble interactions with cells under ultrasound are the key step for the sonoporation process. To date, five types of interactions have been described (Figure 1). Microbubble oscillations near a plasma membrane could leading to a cell “massage”, the “push and pull” phenomena [25, 26] and microbubble jetting through the plasma membrane [27]. This latter is less likely to be the dominant mechanism as shown by in silico studies, in vitro by high-speed optical observations, and in cellulo experiments [28]. A microbubble attached to a cell membrane, could also create enough shear to rupture the membrane due to the fluid streaming around the oscillating bubbles. Recently, we have observed a new event: a penetration of microbubble into a cell during sonoporation process under a specific ultrasound setting [29]. The tracking of fluorescent-labeled microbubbles inside cells after sonoporation has proved this phenomenon. Figure 2 shows two microbubbles (black circle) entering into a cell during ultrasound stimulation, one microbubble was pushed 5 toward the cell deforming the plasma membrane before its cellular penetration. While entering, the microbubble underwent shrinking. This phenomenon took place 6.2 seconds after ultrasound stimulation and 850 ms later, microbubble penetrated inside the cell. This event is slow, compared to the other phenomena described above. Indeed, jetting occurs in the millisecond time scale. Fluorescent microbubbles were observed inside the cells after sonoporation proving their penetration (figure 2C, black arrows). However, it is hard to state if the microbubble has only fused with the plasma membrane or if the structure of the microbubble was preserved after entry. Nevertheless, these results reinforce the strategy on using microbubbles as drug or gene carriers. We have also tracked the early interactions between cells and microbubbles at the beginning of the ultrasound stimulation [29]. In this study, we have used several phospholipid-based microbubbles (SonoVue®, MicromarkerTM, Definity®). Before ultrasound stimulation, microbubbles were randomly distributed all around cells. When ultrasound was turned on, microbubbles immediately interacted with each other forming small clusters (Figure 3) as described by Kotopoulis et al. [30]. These clusters could be observed approximately 15 ms after ultrasound application. Interestingly, every cluster present in the field seemed to be attracted to cells and was found at the vicinity of their plasma membrane several seconds after ultrasound stimulation independently of the ultrasound field direction. It is worth noticing that we did not observe such behavior for hard-shelled
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